RATIONALE:Restrictive allograft syndrome (RAS) is a major cause of mortality following lung transplantation due to progressive fibrosis of the lung allograft with no therapeutic options. Knowledge of the cellular and molecular mechanisms driving fibrosis in RAS remains limited. OBJECTIVE:To characterize the cellular and molecular changes in human RAS lungs through single-cell transcriptomic profiling. METHODS:Single-nucleus RNA-sequencing (snRNA-seq) was performed in peripheral lung tissues from 15 RAS patients undergoing lung re-transplantation, and from 9 healthy control lungs. Findings were validated and extended using histologic techniques including immunofluorescence, RNA in situ hybridization, Elastica-van-Gieson immunohistochemistry, quantitative histological analyses, and micro-CT scans. MEASUREMENTS AND MAIN RESULTS:snRNA-seq analysis of RAS lungs revealed previously undescribed aberrant basaloid cells, ectopic COL15A1+ peribronchial vascular endothelial cells (pVECs), and CTHRC1+ fibrotic fibroblasts. Histologic stains disclosed distinctive distribution patterns: aberrant basaloid cells, primarily localized at the fibrotic edge, together with juxtaposed CTHRC1+ fibrotic fibroblasts and ectopic COL15A1+ pVECs form the fibrotic niche of alveolar fibroelastosis (AFE). PRX+ alveolar microvasculature is partially lost in AFE areas. Micro-CT scans revealed changes from pulmonary to systemic perfusion, facilitated by COL15A1+ pVECs. Last, our data reveals potential therapeutic targets in RAS, including integrin αvβ6, activator of TGFβ. CONCLUSION:Considering the multifaceted differences of RAS and idiopathic pulmonary fibrosis, we revealed a surprising general principle of an entity-spanning composition of the fibrotic niche by aberrant basaloid cells localized at the fibrotic edge, ectopic COL15A1+ pVECs and CTHRC1+ fibrotic fibroblasts. This suggests a flexible but cellular pathogenesis-guided transferability of potential therapeutic approaches between progressive fibrotic lung diseases.

Ultra-high-resolution propagation-based synchrotron phase-contrast CT is an emerging technique for lung imaging. However, its feasibility and diagnostic potential at radiation doses comparable to those used in standard clinical procedures has yet to be established. This study aims to evaluate the performance of phase-contrast CT in comparison with state-of-the-art high-resolution multislice CT and bronchoscopy, and to validate its diagnostic accuracy histologically using porcine and, for the first time, human lung specimens. Phase-contrast CT experiments were conducted at the Italian synchrotron using lung specimens mounted in a custom-made anthropomorphic chest phantom. Imaging utilized two photon-counting detectors under various acquisition settings, followed by artificial intelligence-based denoising. Sequential imaging by phase-contrast CT, multislice CT, and bronchoscopy was performed prior to formaldehyde vapor fixation and histological dissection. Image quality was assessed quantitatively (contrast-to-noise ratio, edge sharpness, power spectra) and qualitatively via radiological scoring across 14 criteria. Phase-contrast CT achieved effective pixel sizes of 0.067 mm (Hydra detector) and 0.038 mm (LAMBDA detector), at radiation doses near full-dose multislice CT (≈ 12 mGy). Denoising improved contrast without major loss of edge sharpness. Radiological scoring showed phase-contrast CT outperformed multislice CT in visualizing peripheral airways and fine parenchymal structures. Histological validation confirmed imaging accuracy. Limitations from source spot size (≈ 200 μm) were noted but did not prevent significant diagnostic improvements. Phase-contrast CT, combined with artificial intelligence-based denoising, offers detailed, non-invasive imaging of lung microstructures at clinically relevant radiation doses. It complements multislice CT, holds potential for clinical adoption in advanced pulmonary diagnostics, and may reduce reliance on invasive biopsies.

Abstract Background Pleuroparenchymal fibroelastosis (PPFE) is a rare, fibrotic lung disease with poor prognosis, usually affecting adults which most commonly occurs idiopathically. Biallelic pathogenic variants in DGUOK cause mitochondrial DNA (mtDNA) depletion syndrome, predominantly affecting infants with severe hepatic and neurological symptoms. Detailed description of pulmonary manifestations with late-onset presentation have not been reported. Methods We describe nine patients with PPFE and DGUOK -associated mitochondriopathy. Clinical, radiological, histopathological, and genetic data were systematically collected from all patients. Functional studies, single nucleus RNA sequencing (snRNAseq), immunofluorescence staining, transmission electron microscopy and respiratory chain enzyme activity assays were conducted on patient-derived fibroblasts, muscle or lung tissues. mtDNA content quantification was performed on whole genome sequencing (WGS) data. Results All patients (ages 5–36) presented with progressive dyspnea, weight loss and some with spontaneous pneumothoraces. Chest computed tomography and lung biopsies showed features of PPFE. Biallelic pathogenic DGUOK variants were identified in all patients, seven of them carry an unreported intronic variant leading to mtDNA depletion. snRNAseq of lung tissue from four pediatric patients identified Aberrant Basaloid cells and intermediate cells as their precursor localized at the fibrotic edge. Mitochondrial alterations were identified by electron microscopy. Conclusion PPFE in children and young adults is associated with DGUOK -related mitochondriopathy. For the first time, we demonstrate Aberrant Basaloid cells in pediatric fibrotic lung tissue. Since pulmonary involvement may be underrecognized or misinterpreted and the clinical presentation may not always be typical of a mitochondriopathy, we recommend genetic testing in all patients with PPFE of unknown origin.

Background:Fibroblasts are important contributors to collagen deposition in interstitial lung diseases (ILD), but their inflammatory role in acute exacerbation of ILD including idiopathic pulmonary fibrosis is currently undefined. Methods:Using a well-described model of Streptococcus pneumoniae-induced exacerbation of lung fibrosis in mice, we found that platelet-derived growth factor receptor A-positive lung fibroblasts developed an early inflammatory phenotype upon infection, including increased production of serum amyloid A (SAA) proteins, as determined by RNA sequencing, single-cell RNA sequencing and secretome profiling of sorted lung fibroblasts. Results:Lung fibroblasts responded to pneumococcal challenge with increased SAA mRNA and protein levels in vitro. SAA levels were significantly elevated in bronchoalveolar lavage (BAL) and plasma of mice with S. pneumoniae-induced exacerbation of adenoviral vector delivery of active transforming growth factor-β1- or bleomycin-induced lung fibrosis. Antibiotic therapy caused a significant drop in SAA levels in BAL and plasma of mice with infection-driven fibrosis exacerbation. Finally, levels of SAA protein were dramatically increased in plasma of patients with acute exacerbation of ILD, but not in patients without exacerbation. Conclusion:Analysis of plasma levels of SAA protein may aid in the identification of underlying inflammation in patients with ILD who are more likely to deteriorate towards a critical clinical stage during acute exacerbation.

Abstract Background Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II-associated features, enable cryopreservation, and capture disease-associated metabolic alterations. Methods HT-II-280+ AT-II cells were isolated by magnetic bead sorting from 63 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 22 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II-associated features were assessed by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability and organoid-forming capacity. Metabolic profiling was performed using extracellular flux assays. Results AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (> 90% in P1), especially in AT-II cells from fibrotic explants. Primary tissue-derived lung organoids display heterogeneous morphologies and sizes, most prominently in fibrotic-derived cultures, as indicated by histology and microcomputed tomography. Culture conditions were optimized to minimize differentiation towards AT-I cells or dedifferentiated epithelial states with partial basaloid features. Expression of key AT-II-associated markers (proSP-C, HT-II-280), and the presence of lamellar bodies were maintained across passages at the population level. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, enabling long-term storage. Fibrotic organoids exhibited disease-associated metabolic reprogramming characterized by a pronounced glycolytic shift with increased ATP production. Conclusion We established a reproducible cell-line-free 3D culture system from primary human AT-II cells of end-stage ILD lungs to generate patient-derived lung organoids. These organoids maintain AT-II-associated features across passages, remain viable after cryostorage, and capture disease-associated metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting increased energy demand. This scalable model in vitro provides a defined resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for future precision medicine applications.

Pleuroparenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease (ILD) with defining histology of intra-alveolar fibrosis with septal elastosis (AFE), suggesting unique cellular disease processes. Here, we present a binational single-nucleus RNA sequencing atlas of PPFE, based on explanted lungs from 40 patients. Immunofluorescence microscopy, RNA in situ hybridization, micro-computed tomography (CT), and hierarchical phase-contrast (HiP) synchrotron CT provided spatial context. We identify PPFE-associated adventitial and elastofibrotic fibroblasts as key drivers of elastotic remodeling within an inflammatory microenvironment, maintained by immune cells forming tertiary lymphoid structures. Spatial mapping reveals an intriguing zonation of AFE, maintained by intercellular circuits between PPFE-associated cell types. Comparative analysis with idiopathic pulmonary fibrosis highlights CTHRC1+ fibrotic fibroblasts and aberrant basaloid cells as conserved profibrotic cellular machinery mediating collagen deposition across ILDs. This integrative atlas defines the cellular landscape of PPFE and dissects elastotic from fibrotic remodeling, providing a molecular rationale for niche-specific therapeutic strategies.

TGF-β1-induced lung injury initially results in surfactant dysfunction and alveolar instability (microatelectases). Morphology showed that at end-expiratory pressure of 2cmH2O the burden of microatelectasis was high but could be mitigated with a pressure of 8cmH2O by 35% without undue strain. Hence, we hypothesized that ventilation of lungs with a higher burden of microatelectasis at positive end-expiratory pressure (PEEP) of 2cmH2O triggers more pronounced injury progression compared to PEEP=8cmH2O. Mice were randomized to receive either TGF-β1 (AdTGF-β1) for injury induction or empty control vector (AdCl). After a second randomization one week later, mice were ventilated for 4 h with PEEP = 2 or 8cmH2O resulting in 4 experimental groups: AdTGF-β1-PEEP2 (n = 11), AdTGF-β1-PEEP8 (n = 10), AdCl-PEEP2 (n = 11) and AdCl-PEEP8 (n = 10). During ventilation, every 30 min, Quick-Prime (tissue elastance) and Snap-Shot (dynamic compliance) measurements were performed immediately before and after deep inflations. Finally, lungs were either fixed for stereology (both PEEP2 groups n = 6/ both PEEP 8 groups n = 5) or broncho-alveolar lavage (n = 5) and tissue harvest for transcriptome analyses. During ventilation the increase in tissue elastance was largest in AdTGF-β1-PEEP2 group but reversible by deep inflations. Finally, both AdTGF-β1-groups showed comparable worsening in lung mechanics, and BAL-albumin and neutrophils were elevated. Quantitative morphology showed no differences and transcriptome minimal differences attributable to PEEP-level. Low-PEEP ventilation of AdTGF-β1 lungs with a high burden of microatelectases induced lung mechanical worsening most likely due to progressive alveolar derecruitment. This injury pattern was reversible upon deep inflations and not linked with signs of persistent lung injury such as vascular leakage, edema or inflammation.

[This corrects the article DOI: 10.1371/journal.pone.0297889.].

Background Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II identity, enable cryopreservation, and capture disease-specific metabolic alterations. Methods HT-II-280 + AT-II cells were isolated by magnetic bead sorting from 62 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 21 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II identity was verified by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability. Metabolic profiling was performed using extracellular flux assays. Results AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (> 90% in P1), especially in AT-II cells from fibrotic explants. Interestingly, primary tissue-derived lung organoids display heterogeneous morphologies and sizes, particularly in fibrotic-derived cultures indicated by histology and microcomputed tomography. Culturing conditions were optimized to avoid differentiation towards AT-I cells or aberrant basaloid cells. Lineage fidelity was preserved across passages, with stable expression of proSP-C, HT-II-280, and pronounced presence of lamellar bodies. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, highlighting possibility for on demand long-term storage. Fibrotic organoids exhibited metabolic reprogramming illustrated by a pronounced glycolytic shift with increased ATP production. Conclusion We established a robust and reproducible cell-line-free 3D platform from primary human AT-II cells of end-stage ILD lungs to generate personalized lung organoids. These organoids retain AT-II identity across passages, remain viable after cryostorage, and recapitulate patient-specific metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting possible mitochondrial dysfunction and high energy demand. This reproducible scalable model provides a transferable resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for precision medicine.

Surfactant protein B (SP-B) is essential for surface tension reducing function of pulmonary surfactant and alveolar unfolding processes during inspiration. SP-B is reduced early in acute lung injury. Hence, we hypothesize that 1) reduced SP-B expression increases susceptibility to ventilation-induced lung injury (VILI), and 2) deep inflations (DI) are protective against VILI. Conditional SP-B knockout mice were randomized into OFF (reduced SP-B) and ON groups (normal SP-B) and subjected to mechanical ventilation at zero end-expiratory pressure. Over 4 h of ventilation, either 4 or 16 DI were administered. Lung mechanics were recorded, and pulmonary structure was quantified by design-based stereology. Inflammatory cells and bulk RNA sequencing were measured in bronchoalveolar lavage (BAL) and tissue, respectively. No differences in inflammatory cells in BAL were detected between ON and OFF groups. During ventilation, alveolar derecruitment-related increase in elastance was most pronounced in OFF-4DI but reversible by DI so that lung mechanics did not worsen. Finally, volumes of the alveolar liquid lining layer and the intracellular surfactant were largest, whereas the surface area of the apical plasma membrane of type II pneumocytes was smallest in OFF-4DI, suggesting impaired surfactant secretion. A higher frequency of DI prevented these abnormalities. Electron microscopy revealed disorganized tight junctions between alveolar epithelial cells in OFF-4DI, which was linked with decreased expression of genes relevant to the apical junctional complex. Reduced SP-B resulted in a progressive increase in surface tension and a disturbed fluid balance without triggering definite VILI. Maintenance of residual surfactant function is highly dependent on DI in conditions of reduced SP-B levels. NEW & NOTEWORTHY Surfactant protein B (SP-B) is critical for efficient surfactant function in the lung. Reduced SP-B levels occur at an early stage of acute lung injury and impair alveolar unfolding. In this study, we demonstrate that mechanical ventilation of lungs with reduced SP-B levels does not trigger ventilation-induced lung injury but results in disbalance of alveolar fluid volume and increase in surface tension due to failure of surfactant maintenance. Deep inflations prevent these ventilation-induced effects.

Surfactant protein B (SP-B) contributes to surface tension reduction at the pulmonary air-liquid interface. In lung injury, downregulation of alveolar SP-B is an early finding. During inspiration, unfolding processes of the interalveolar septa are considered as physiological micromechanical mechanisms that might become injurious in the presence of high surface tension, thus propagating injury. The aim of the present study was to quantify SP-B deficiency-related alterations in micromechanics of the blood-gas-barrier (BGB) during inspiration at physiological lung volumes. Our transgenic mouse line expressed SP-B under control of a doxycycline-dependent promoter. Two days after withdrawal of doxycycline (Dox-off), the mean SP-B level declined by 86%. In Dox-on and Dox-off groups, lung mechanics were assessed before the lungs were fixed at increasing airway opening pressures on inspiration or subjected to broncho-alveolar lavage (BAL) and gene expression analyses. Fixed lungs were investigated by design-based stereology. In Dox-off the BAL-albumin, alveolar hypophase volume and tissue elastance slightly increased concomitantly with proinflammatory gene-expression profiles while inflammatory cells remained unchanged. Stereology demonstrated increased derecruited septa and folded BGB in Dox-off. Although in Dox-on inspiratory unfolding of the BGB resulted in an increase of air-exposed alveolar epithelium, this mechanism was not prevalent in Dox-off where, instead, dilation of acinar airspaces occurred (mainly in the alveolar duct compartment). Inspiratory increases in surface area of the epithelial basal lamina were absent in both groups. In essence, while stretching was not observed in any group, inspiratory unfolding of the BGB is a dominant mechanism in healthy lungs but absent in SP-B deficiency.NEW & NOTEWORTHY Surfactant protein B (SP-B) deficiency is an early feature in experimental and clinical acute lung injury and might contribute to injury progression via mechanical stress of the blood-gas barrier (BGB) even at physiological lung volumes. Although in healthy mice, inspiratory micromechanics was dominated by unfolding processes of the BGB, this mechanism was absent in SP-B deficiency. Instead interalveolar septa appeared to be stiffened, resulting in a dilation of alveolar ducts. Stretching of the BGB was not found.

OBJECTIVE:Rats are born with morphologically immature lungs, but intact surfactant system. The aim of this study was to characterize the surfactant producing alveolar epithelial cells type II (AEII) during alveolarization and find relationships between the intracellular surfactant pool and alveolar surface area, lung volume and body weight. METHODS:After exsanguination, lungs of 3, 7, 14, 21 and 90 days old rats were inflated with a pressure of 10 mm H20 and fixed by perfusion and prepared for light and electron microscopy. Using different stereological parameters AEII were characterized. RESULTS:At day 21, the end of bulk alveolarization, the alveolar surface and the number of AEII increased significantly but their volume and size did not change compared to values before alveolarization. The number of AEII, but not the AEII volume correlated significantly with alveolar surface and lung volume. The size and volume weighted mean volume of lamellar bodies (Lb) as well as the Lb volume per AEII did not change during alveolarization. Total Lb volume was significantly higher at the end of bulk alveolarization compared to values before alveolarization. CONCLUSION:The adaptation of the intracellular surfactant during postnatal development occurred predominantly by increasing the number of AEII.

RATIONALE: The entity pleuro-parenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease histologically characterized by the pattern of alveolar fibroelastosis with distinct hallmarks in comparison to other fibrosing lung diseases. Driver cells in the pathogenetic cascade remain incompletely understood. METHODS: Fresh lung tissue from 3 PPFE explants and from 8 control patients within distinct non-PPFE interstitial lung disease (ILD) was collected. Correlative light- and electron microscopy (CLEM) was performed to characterize mesenchymal cells in the area of active fibrotic remodelling. Mesenchymal cell outgrow of PPFE and control tissues were cultured and analysed by scanning electron microscopy (SEM) and single-cell RNA sequencing (scRNAseq). Celltype annotation was performed by reference mapping to recently generated snRNAseq data from a larger PPFE-cohort. RESULTS: CLEM analysis of the active subpleural remodelling zone, revealed a network of numerous elongated myofibroblast-like cells characterized by significant cellular extensions in association with elastic fibres and collagen matrices. Furthermore CLEM identified light microscopic intra-fibrotic fibrils as infiltrating cell body extensions, phenotypically resembling adventitial fibroblasts. Cell outgrow experiments identified a PPFE-specific cell population showing overlap with features of adventitial fibroblast (MFAP5+, CEMIP+) and myofibroblasts (CTHRC1+). Morphology-wise, SEM revealed numerous elongated cell extensions within the outgrown PPFE mesenchyme. KNN mapping to a reference snRNAseq dataset of a larger multinational PPFE cohort identified the outgrown PPFE-specific cells as a subpopulation of adventitial fibroblasts and myofibroblasts. CONCLUSIONS: CLEM and scRNAseq analysis shed light on an invasive mesenchymal cell population in PPFE with transcriptomic and ultrastructural features of adventitial fibroblast, which may be a driver of fibroelastotic remodeling in PPFE.

RATIONALE: Pleuroparenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease, histologically characterized by alveolar fibroelastosis (AFE) with comparably high prevalence in females. Currently, there are no effective antiinflammatory or antifibrotic treatments available, with lung transplantation being the only option for advanced disease. There is a critical need to understand the disease's pathogenesis at a single-cell resolution to identify novel treatment targets. METHODS: Formalin-fixed paraffin-embedded tissues from explant lungs of a German (24 PPFE patients and 16 downsizing/tumor-free resection controls) and a French cohort (17 PPFE patients) were stained (Elastica van Gieson). Specimen with pathologist confirmed AFE pattern were selected and analyzed using single-nuclear sequencing (snRNAseq). Selected specimen underwent micro-CT scanning beforehand. Identified cell populations were localized through immunofluorescence (IF) and multiplex RNA in-situ hybridization. RESULTS: snRNAseq revealed aberrant basaloid epithelial cells, localized as KRT17+, PTGS2+ and CTSE+ positive cells at the edge between subpleural AFE lesions and intact alveolar parenchyma. Comparative K-nearest neighbors projection on snRNAseq data of IPF patients (unpublished) analyzing 1108 common expressed genes confirmed the aberrant basaloid cell phenotype in the PPFE epithelium. Immunofluorescence revealed a homogenous cellular architecture of the fibroelastotic edge of a) aberrant basaloid cells (intraepithelial cell frequencies in German PPFE: 4.2% ± 0.03%; French PPFE: 4.6% ± 0.04%; Control 0.1 ± 0.01%) followed by b) CTHRC1+ myofibroblasts and c) Tertiary lymphoid follicles which organize continuously along the fibroelastotic-remodeling edge. They are composed of CD20+ B-cells, CD4+ or CD8+ T-cells. Analogous snRNAseq analysis of the immune cell lineage yielded a concordant increase in the respective lymphocyte subpopulations. Analysis of the mesenchymal lineage confirmed elevated counts of CTHRC1+ myofibroblasts, but also in MFAP5+, SFRP1+ and PI16+ positive adventitial fibroblasts among both investigated PPFE cohorts. Changes in the endothelial compartment included the loss of PRX+ aerocytes, emergence of COL15A1+ systemic venous, in addition to a marked expansion of lymphatic endothelial cells, which located in the subpleural AFE lesion. 3D micro-CT indicated aberrant neovascularization originating from the pleura. CONCLUSIONS: The study highlights a pathogenic tetrad of aberrant basaloid epithelial dedifferentiation, adventitial (myo-)fibroblast driven fibroelastosis and lymphatic neovascularization, paralleled by the emergence of lymphocytic cell infiltrates forming tertiary lymphoid structures. The surprising findings within the PPFE atlas will serve as a rationale for novel therapeutic approaches for this hitherto incurable disease.

A minimal diffusion barrier is key to the pulmonary gas exchange. In alveolar capillary dysplasia (ACD), a rare genetically driven disease of early infancy, this crucial fibrovascular interface is compromised while the underlying pathophysiology is insufficiently understood. Recent in-depth analyses of vascular alterations in adult lung disease encouraged researchers to extend these studies to ACD and compare the changes of the microvasculature. Lung tissue samples of children with ACD (n = 12), adults with non-specific interstitial pneumonia (n = 12), and controls (n = 20) were studied using transmission electron microscopy, single -gene sequencing, immunostaining, exome sequencing, and broad transcriptome profiling. In ACD, pulmonary capillary basement membranes were hypertrophied, thickened, and multilamellated. Transcriptome profiling revealed increased CDH5, COL4A1, COL15A1, PTK2B, and FN1 and decreased VIT expression, confirmed by immunohistochemistry. In contrast, non-specific interstitial pneumonia samples showed a regular basement membrane architecture with preserved VIT expression but also increased COL15A1 thorn vessels. This study provides insight into the ultrastructure and pathophysiology of ACD. The lack of normally developed lung capillaries appeared to cause a replacement by COL15A1 thorn vessels, a mechanism recently described in interstitial lung disease. The VIT loss and FN1 overexpression might contribute to the unique appearance of basement membranes in ACD. Future studies are needed to explore the therapeutic potential of down -regulating the expression of FN1 and balancing VIT deficiency. (Am J Pathol 2024, 194: 180-194; https://doi.org/10.1016/ j.ajpath.2023.10.012)